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Journal of Virology, August 1999, p. 6293-6298, Vol. 73, No. 8
Centro Nacional de Biología
Fundamental,
Received 17 March 1999/Accepted 10 May 1999
Nonconservative substitutions for Tyr-115 in the reverse
transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) lead
to enzymes displaying lower affinity for deoxynucleoside triphosphates
(dNTPs) (A. M. Martín-Hernández, E. Domingo, and L. Menéndez-Arias, EMBO J. 15:4434-4442, 1996). Several mutations at this position (Y115W, Y115L, Y115A, and Y115D) were introduced in an
infectious HIV-1 clone, and the replicative capacity of the mutant
viruses was monitored. Y115W was the only mutant able to replicate in
MT-4 cells, albeit very poorly. Nucleotide sequence analysis of the
progeny virus recovered from supernatants of four independent
transfection experiments showed that the Y115W mutation was maintained.
However, in all cases an additional substitution in the primer grip of
the RT (M230I) emerged when the virus increased its replication
capacity. Using recombinant HIV-1 RT, we demonstrate that M230I
mitigates the polymerase activity defect of the Y115W mutant, by
increasing the dNTP binding affinity of the enzyme. The second-site
suppressor effects observed were mediated by mutations in the 66-kDa
subunit of the RT, as demonstrated with chimeric heterodimers.
Examination of available crystal structures of HIV-1 RT suggests a
possible mechanism for restoration of enzyme activity by the
second-site revertant.
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Second-Site Reversion of a Human Immunodeficiency
Virus Type 1 Reverse Transcriptase Mutant That Restores Enzyme
Function and Replication Capacity
*
Corresponding author. Mailing address: Centro de
Biología Molecular "Severo Ochoa," Consejo Superior de
Investigaciones Científicas-Universidad Autónoma de
Madrid, Cantoblanco, 28049 Madrid, Spain. Phone: 34-91-3978477. Fax: 34-91-3974799. E-mail: LMENENDEZ{at}CBM.UAM.ES.
Journal of Virology, August 1999, p. 6293-6298, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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